Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering

Area of Science:

• Microbial biotechnology
• Metabolic engineering
• Biopolymer production

Background:

• Poly-γ-glutamic acid (γ-PGA) is a versatile biopolymer with applications in medicine, food, and environmental remediation.
• Enhancing γ-PGA production in microbial hosts like Bacillus amyloliquefaciens is crucial for cost-effective industrial applications.
• Previous metabolic engineering efforts have shown potential but require systematic approaches for significant yield improvements.

Purpose of the Study:

• To systematically engineer the metabolic networks of Bacillus amyloliquefaciens for enhanced poly-γ-glutamic acid (γ-PGA) production.
• To identify and manipulate key genes involved in γ-PGA synthesis, degradation, precursor supply, and byproduct formation.
• To achieve a significant increase in γ-PGA titer through a multi-step metabolic engineering strategy.

Main Methods:

• Gene deletions were performed in Bacillus amyloliquefaciens to eliminate pathways for byproduct synthesis (epsA-O, sac, lps, pta) and γ-PGA degradation (pgdS, cwlO).
• The luxS gene involved in autoinducer synthesis was deleted, and the expression of glutamate dehydrogenase (rocG) and glutamine synthetase (glnA) genes was repressed using synthetic small regulatory RNAs.
• Fed-batch cultivation was employed to optimize the production conditions for the engineered strain.

Main Results:

• Deletion of byproduct synthesis genes increased γ-PGA purity and slightly improved titer (3.8 to 4.15 g/L).
• Subsequent deletion of γ-PGA degradation genes significantly increased production (4.15 to 9.18 g/L).
• Repression of rocG led to the highest γ-PGA titer (11.04 g/L in flasks, 20.3 g/L in fed-batch), a 5.34-fold increase over the wild-type strain.

Conclusions:

• A systematic metabolic engineering approach successfully enhanced γ-PGA production in Bacillus amyloliquefaciens.
• The developed engineering strategies provide a valuable framework for improving the production of γ-PGA and other valuable metabolites.
• The engineered strain demonstrates significant potential for industrial-scale biopolymer production.